We hypothesize that in sickle cell anemia, the source of hemodynamic abnormalities is not only abnormal red cell rheology (sickling and adhesion to endothelium), but a derangement of microvascular controls secondary to endothelial dysfunction. This proposal focuses on two potential modulators of microvascular flow and vasoocclusion in sickle cell anemia. First, endothelial dysfunction in response to hypoxia, mechanical injury (red cell sickling and adhesion), and oxidative stress (secondary to transient ischemic episodes) would affect the ability of microvasculature to respond to rheological challenge. Second, abnormal adherence of red cells to vascular endothelium would not only result in endothelial injury, but also in a prosickling environment. The objective of this revised proposal is to examine the relationship between abnormal rheology, oxidative stress and vascular tone under in vivo conditions in the sickle context, using transgenic and knockout sickle mouse lines. We will test the following specific hypotheses: 1) Test the hypothesis that sickling and attendant flow abnormalities (e.g., transient vasoocclusive events) will cause oxidative stress, microvascular injury and vascular tone abnormalities. To test this hypothesis, we will investigate the effect of enhanced sickling (hypoxia), examine the effect of arginine supplementation, and evaluate the effect of selected anti-oxidants; 2) Test the hypothesis that genetic and experimental modulations of red cell density and polymer formation will impact endothelium and thereby microvascular function. To test this aspect, we will determine the effects of anti-sickling fetal hemoglobin and experimental modulations of red cell density; 3) Test the hypothesis that red cell adhesion in vivo not only contributes to endothelial injury but plays a crucial role in microvascular obstruction. To test this hypothesis, we will focus on the role of endothelial activation, specific adhesion molecules, hypoxia, NO, anti-oxidants, and use DNA microarray technology to identify genes regulated by adhesion inducing factors. The proposed research involves participation of scientists with expertise in microcirculation, hematology, biochemistry, cell biology and molecular biology. These studies are expected to elucidate new mechanisms with relevance to human sickle cell disease and with potential therapeutic implications. [unreadable] [unreadable]